Drug resistant TB: Types, prevalence, threat & detection

The Acquired Immune Deficiency Syndrome, Malaria and Tuberculosis together rank highest in the list of threatening infectious diseases in the world, especially in the middle- and low-income group countries. Human tuberculosis caused by Mycobacterium tuberculosis or MTB is an airborne infectious disease infecting nearly one third of the world population and resulting in around 1.6 million deaths
annually.

Treatment of tuberculosis has assumed natural priority across the world and over twenty different drugs have been developed. These anti tubercular drugs are administered in different combinations and under different circumstances. For example, a set of drugs are administered as first line treatment and are intended for patients who are infected with the pathogen that is sensitive to all kind of drugs. These four first line drugs comprising of rifampicin, isoniazid, pyrazinamide and ethambutol form the core protocol for treatment that last for six to nine months.

The resistance to drugs by Mycobacterium tuberculosis is one of the primary contributors of antimicrobial resistance across the world and also a serious public health threat. In a year, around half a million people fall prey to drug resistant tuberculosis bacteria. The consequences of such infection are heavy. The treatment is harder, longer and very often with comparatively poorer clinical outcomes.  The economic and social fallouts are equally significant. Only a fraction of such patients gets quality healthcare attention and a very large number of them go undiagnosed and are potential source of lateral infection posing a serious challenge to tuberculosis control programmes operational in the country.

There are multiple reasons for tuberculosis drugs to fail in an infected patient the most significant of them being evolution of drug resistant Mycobacterium tuberculosis strains in the population.

There are several types of drug resistant Mycobacterium tuberculosis. Multidrug-resistant TB (MDR TB) is caused by tuberculosis bacteria that are resistant to at least isoniazid and rifampin, the two most potent anti tuberculosis drugs. These drugs are used for the treatment of all patients suffering from tuberculosis disease.

The next level of drug resistance is represented by the Pre-Extensively Drug-resistant TB (pre-XDR TB). This is a specific category of MDR TB and is represented by those MDR variants which are additionally resistant to either a fluroquinolone or a second line injectable such as amikacin, capreomycin, and kanamycin apart from the original resistance acquired against isoniazid and rifampicin.

The Extensively drug-resistant TB (XDR TB) is relatively rare in the world population as on this day. Such strains are resistant to isoniazid and rifampin, a fluroquinolone, as well as a second-line injectable (amikacin, capreomycin, and kanamycin) or those that are resistant to isoniazid, rifampin, a fluroquinolone, and two of the latest and new generation anti tuberculosis drug, namely bedaquiline or linezolid. Needless to say, in cases of XDR TB infection, the treatment options are far less in availability and efficacy both.

Four first line anti tuberculosis drugs, namely rifampicin, isoniazid, streptomycin and ethambutanol together play the role of a double-edged sword. While on one hand they effectively kill pathogenic Mycobacterium tuberculosis but on the other, they assist in development of drug resistant strains that defy these very drugs that are designed to kill them.

Multi drug resistant Mycobacterium tuberculosis acquires the capability to resist an anti-tubercular drug mainly by mutations within critical genes present in its genome. Some of these genes include rpoB, inhA, katG, rpsL, and embB respectively. Typically, occurrence of point mutation within these genes dictates multi drug resistance. These mutations are single base pair change and have a profound effect on the way Mycobacterium tuberculosis responds to anti tubercular drugs. Similar mutation in other genes linked to other different anti tubercular drugs result in occurrence of pre-XDR TB and XDR TB. Thus, detecting such point mutations hold key to diagnosis of all forms of
drug resistant Mycobacterium tuberculosis.

A large number of methods and techniques evolved for detecting point mutation within gene of interest in drug resistant Mycobacterium tuberculosis. While culture-based methods are the most reliable, they take significant amount of time apart from being expensive, thus hampering timely and early treatment. A large number of ‘probes’-based methods developed over the time, key among them being the line probe assays (LPA), GeneXpert (Cepheid Inc., Sunnyvale, CA, USA) and a vast array of home-grown as well as commercial detection solutions running on end point and real time thermal cycling platforms.

In recent times, NGS or the Next Generation Sequencing has evolved that can provide rapid and detailed information of the Mycobacterium tuberculosis genome through massive parallel sequencing. While the advantages are many that include detection of de novo mutations within the Mycobacterium tuberculosis genome, the key disadvantages include comparatively high cost of test, poor sensitivity while using sputum rather than culture isolate, heavy investment that often run into several crores of Indian rupee and need for highly specialized staffs. Low number of sample-processing NGS platforms deliver high-cost reports while high number of sample-processing NGS platforms provide low-cost reports but in lieu of the need for a large volume of samples which bring in the risk of high turnaround time in reporting if desired run-quorum is not met every time.

Drug resistant Mycobacterium tuberculosis gradually evolve in a human system and grow in number before dominating the bio-flora. Once domination is achieved, it spreads laterally and horizontally, infecting fresh population and the newly infected individuals soon defy treatment regimens. This points at the need for an ultra-early detection technology for drug resistant Mycobacterium tuberculosis variants that can detect the evolving mutants much early in a clinical sample and allow early isolation of patients within the society to prevent harmful lateral infection to others.

It may be noted that most Mycobacterium tuberculosis drug resistant detection technology rely on dual-labelled probes or hybridization probes that function by reading cumulative fluorescence generated from the probes as the reaction progresses. Interestingly, there are no specialized mechanism in place in most of these assays that is specifically dedicated to ultra-sensitive detection of the mutations in a clinical environment. The dual-labelled probes (Taqman probes) are primarily specialized in their ability for allelic discrimination and LPAs rely on hybridization of probe on targets for mutation detection and carry regular quantum of sensitivity that is typically associated with any probe-target hybridization-based assay. The next generation sequencing or NGS is an exception because the machine generates DNA sequence from virtually every DNA molecule available at its disposal thus naturally enhancing sensitivity. However, the trade-offs are a pain point and by way of the need for a very high infrastructure cost, specialized manpower and comparatively high cost of running.

In this backdrop it is worthy to note that Wobble Base Bioresearch Private Limited, an Indian biotechnology startup recently received the prestigious and highly competitive national research grant under the SBIRI scheme from BIRAC, an industry-academia interface of the Department of Biotechnology, Govt of India. Poised to execute its fifth such grant proposal from BIRAC and BIRAC-involved umbrella programmes in the last 9 years, Wobble Base Bioresearch joins the prestigious league of national research groups entrusted by the Government to develop novel diagnostic solution to diagnose drug resistant Mycobacterium tuberculosis.

For those aspiring to apply for a SBIRI research grant, the selection process may be noted as one of the most stringent in the industry. Post submission, following an area panel review by senior experts, the proposal moves to the desk of technical expert committee which is chaired by eminent scientists of the country. This follows a series of challenging presentations that are carefully evaluated by the technical expert committee before it is recommended for detailed patent search to confirm inadvertent breach of filed patents if any during the proposed course of work. This then moves on to the house of the Apex committee for a final decision and if recommended, financial concurrence and other formalities ensue prior to formal initiation of the project.

Wobble Base Bioresearch is using its innovative molecular allele silencers as potent tool that ‘silences’ the non-mutated genes from participating in the detection reaction thus exposing the mutated ones only which are involved with drug resistance. This dramatically enhances sensitivity of the assay and allow early detection of drug resistant variants of Mycobacterium tuberculosis in a clinical environment.

The theme of World TB Day 2021 was ‘The Clock is Ticking’ and that of 2022, ‘Invest to End TB. Save Lives’. These slogans are to underline and convey the sense of urgency to global technology players that time is running out to collectively eradicate MTB from the world. It now assumes greater significance due to the Covid- 19 pandemic that has already placed the End TB movement at a great risk.  

India launched the ambitious National Strategic Plan for TB Elimination to reach the target of making India TB free by year 2025. This mission will be challenging if detection solution of drug resistant TB is not revisited again with newer methods and approaches that address cost, quality and speed, and effectively control the emergence and spread of drug resistant tuberculosis  in our population.